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, 85 (5), 1225-32

Photocytotoxicity of the Fluorescent Nonsteroidal Androgen Receptor Ligand TDPQ

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Photocytotoxicity of the Fluorescent Nonsteroidal Androgen Receptor Ligand TDPQ

Piotr J Bilski et al. Photochem Photobiol.

Abstract

1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline (TDPQ), a selective nonsteroidal androgen receptor (AR) ligand, is a fluorescent compound. We characterized its spectral properties in comparison with the structural precursor carbostyril 151 (C151) and with its racemic structural isomer 4-ethyl-1,2,3,4-tetrahydro-6-(trifluoromethyl)-8-pyridino[5,6-g]quinoline (ETPQ). The absorption maximum in CH3CN of either TDPQ or ETPQ is 400 nm whereas that of C151 is 350 nm. The fluorescence lifetimes (tau) and quantum yields (phif) in CH3CN are typical of fluorescent dyes: TDPQ (4.2 ns, 0.8) and ETPQ (4.6 ns, 0.76). C151 showed lower tau and phif of 0.2 ns and 0.02, respectively. TDPQ can function as a fluorescent label at (sub)micromolar concentrations. We detected TDPQ fluorescence in human breast tumor cells using confocal microscopy. While the fluorescence maxima of the compounds were solvent insensitive, the phif for ETPQ decreased in aqueous solutions regardless of the presence of albumin or DNA. The phif of TDPQ was less affected. The quantum yield of singlet oxygen (1O2) photosensitization (phiso) by TDPQ and ETPQ was about 7% in CH3CN, sufficient to induce photocytotoxicity. TDPQ was photocytotoxic in AR-positive MDA-MB-453 breast cancer cells but not in AR-negative MDA-MB-231 cells. The combination of AR selectivity with photocytotoxicity makes TDPQ a promising candidate for selective targeting of AR-positive cells during photodynamic therapy.

Figures

Figure 1
Figure 1
Fluorescence (A, C) and absorption (B, D) spectra of TDPQ (100 μM), ETPQ (100 μM), C151 (992 μM) in CH3CN (A, B) and water (C, D), and QS (200 μM) in 1N H2SO4 (A, B). The fluorescence spectrum of C151 in CH3CN has been multiplied 10× for clarity of presentation on the same axes (A). The fluorescence spectra (measured in a 0.5cm path length fluorescence suprasil cell) were corrected and normalized to the same number of absorbed photons. Fluorescence excitation wavelength (λext) shown by scattered light is also indicated. TDPQ, solid black line; ETPQ; dashed red line; C151, solid blue line; QS, dotted green line
Figure 2
Figure 2
Fluorescence (A) and absorption (B) spectra of TDPQ (100 μM), ETPQ (100 μM), and C151 (992 μM) in SDS micelles (5 mM). Complete description and symbols are as shown in Fig. 1.
Figure 3
Figure 3
Fluorescence (A, C) and absorption (B, D) spectra of TDPQ (100 μM), ETPQ (100 μM), and C151 (992 μM) in H2O in the presence of calf thymus DNA (0.5 mg/ml) (A, B), and bovine albumin (0.5 mg/ml) (C, D). Complete description and symbols are as shown in Fig. 1.
Figure 4
Figure 4
1O2 phosphorescence spectra photosensitized at 366 nm by TDPQ and perinaphthenone in CH3CN and by TDPQ in D2O (baseline). The spectrum produced by TDPQ in CH3CN was multiplied 10× for clarity of presentation on the same axes. The quantum yields for 1O2 produced by both TDPQ and ETPQ are also indicated. TDPQ, solid black line; perinaphthenone, solid blue line.
Figure 5
Figure 5
Phase contrast (A, B) and TDPQ confocal fluorescence images (C, D) of AR-negative MDA-MB-231 (left panel) and AR-positive MDA-MB-453 (right panel) human breast cancer cells. Cells were incubated with 100 nM TDPQ for 1 hr at 37 C and subjected to detection of TDPQ fluorescence by using two-photon absorption LSCM. Scale bar, 20 μm.
Figure 6
Figure 6
Detection of AR and TDPQ/light-induced cell death in AR-negative MDA-MB-231 (left panels) and AR-positive MDA-MB-453 (right panels) human breast cancer cells. A, B) Immunocytochemical detection of AR in formalin-fixed, Triton X-100 permeabilized MDA-MB-231 and MDA-MB-453 cells. The cells were incubated with 100 nM TDPQ for 1 hr and subjected to AR immunocytochemistry using PG-21 primary antibody. C, D) Effect of TDPQ and light irradiation on MDA-MB-231 and MDA-MB-453 cell death. Cells were incubated with 300 nM TDPQ for 1hr and irradiated with 12 J/mm2 at 405 nm. Six-hours later, nuclear dyes (PI and Hoechst 33342) were added and images were captured using conventional fluorescence microscopy equipped with appropriate excitation and emission filters for PI (red) or Hoechst 33342 (blue), respectively. Nuclei of all cells are stained blue, while only nuclei of compromised cells undergoing death are stained red. Circles indicate the size of irradiated area. No cell death was observed in non-irradiated cells outside the circled regions. Scale bar for A-B, 20 μm; for C, D, 400 μm.
Scheme 1
Scheme 1
TDPQ: 1,2,3,4-tetrahydro-2,2-dimethyl-6-(trifluoromethyl)-8-pyridono[5,6-g]quinoline ETPQ: 4-ethyl-1,2,3,4-tetrahydro-6-(trifluoromethyl)-8-pyridino[5,6-g]quinoline C151: 7-hydroxy-4-(trifluoromethyl)-2(1H)-quinolinone (Carbostyril 151) *The asterisk in the ETPQ structure denotes an optically active carbon center.

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